Analysis of vitellogenin mRNA by quantitive reverse transcription polymerase chain reaction (RT–PCR) in juvenile fish exposed for 12 months to nonylphenol

Abstract

Nonylphenol (NP) is a degradation product of surfactants that enter aquatic systems mainly via sewage treatment plants. It has been shown that the estrogenic activity of NP results in the induction of vitellogenin (VG) in male fish after short-term exposure, but the effects after long-term exposure to environmentally relevant concentrations remain unclear. Vitellogenin is a precursor of egg yolk proteins, synthesized in the liver under the control of the female sex hormone estradiol and transferred to the ovaries via the blood. Induction of VG can therefore serve as a biomarker for estrogenicity. In this study, rainbow trout eggs were exposed after fertilization to NP concentrations of 1 and 10 μg/l. Exposure occurred throughout the embryonic, larval and juvenile period under controlled laboratory conditions. After 12 months, induction of VG mRNA was analyzed in the liver by quantitative RT–PCR, and VG protein using polyclonal antibodies in Western blots. The development of quantitative RT–PCR included primer design, competitive PCR using heterologous standards and titration. Both VG mRNA and protein were induced in NP-exposed rainbow trout in a dose-dependent manner. In male fish, increases in VG mRNA and protein were already observed at 1 μg/l NP. This study shows that chronic exposure of fish early life stages to environmentally realistic concentrations of NP leads to induction of vitellogenin.